Abstract: Glycoprotein B (gB) of human cytomegalovirus (HCMV) is a major component of the virion envelope and plays an essential role in viral fusion with host cell membranes during early steps of infection in all cell types. gB is highly immunogenic and antibodies directed at gB neutralize virus infectivity in all cell types that have been studied. Clinical trials that have utilized recombinant gB as a prophylactic vaccine have provided equivocal evidence of efficacy, likely secondary to the observation that gB in these preparations are in a post-fusion conformation. Similarly, virus neutralizing human monoclonal antibodies (Mabs) reactive with gB presumably recognize both pre- and postfusion conformations of gB. Mabs specific for prefusion conformers of gB have not been described nor is the structure of this conformation of gB known. In this proposal we will generate human and murine Mabs specific to pre-fusion conformers of gB by using a novel immunization strategy and stringent screening of antibody producing clones. These reagents will be combined with existing gB specific Mabs and gB constructs, including polypeptides expressing discontinuous epitopes, to map antigenic structures specific for the prefusion conformation of gB. Together this data will be used to further refine the structure of gB and generate models of prefusion conformers of gB that can be experimentally validated. Results from these studies can be expected to advance both HCMV vaccine design and the production of therapeutic Mabs.